Plasmid combination and application thereof in preparing modified immune cells

ABSTRACT

Provided in the present disclosure is a method for using a four-plasmid system to prepare modified immune effector cells. The method comprises: forming a lentivirus by using four plasmids within 293T cells, extracting and obtaining the lentivirus, then transfecting immune effector cells by using the lentivirus, and expressing a chimeric antigen receptor. Also provided in the present disclosure is a use of the immune effector cell obtained by using the described method and of a composition containing the immune effector cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 17/412,617,filed on Aug. 26, 2021, which is a continuation of InternationalApplication No. PCT/CN2020/136542, filed on Dec. 15, 2020, which claimspriority of Chinese Patent Application No. CN 201911301518.8, filed onDec. 17, 2019, of Chinese Patent Application No. CN 202011274810.8,filed on Nov. 16, 2020, and of Chinese Patent Application No. CN202011433671.9, filed on Dec. 10, 2020. The contents of each of theseapplications are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The sequence listing provided in the file named SL15829-0001-01000.xmlwith a size of 5,664 bytes, which was created on Sep. 30, 2022 and filedon Oct. 5, 2022, is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to the field of biomedicine. Morespecifically, the present disclosure relates to a method fortransfecting 293T cells using a four-plasmid system, comprising forminga lentivirus by using four plasmids within 293T cells, extracting andobtaining the lentivirus, and then transfecting the T cells with thelentivirus to express a chimera antigen receptor targeting CD19. Thepresent disclosure further relates to an immune effector cell obtainedby using the method, a composition comprising the immune effector cell,and use thereof.

BACKGROUND OF THE INVENTION

At present, clinical treatment for acute lymphoblastic leukemia (ALL),chronic lymphocytic leukemia (CLL) and B-cell lymphoma mainly includeschemotherapy, stem cell transplantation, and biological therapy.Although such therapies can achieve certain efficacy, relapsed orrefractory leukemia still remains as a major issue difficult to betackled. As a new treatment strategy, cellular immunotherapy of tumorhas become a hot topic in recent researches. CD19 is widely expressed onthe surface of almost all the B-cell tumor cells, while being rarelyexpressed in other parenchymal cells and hematopoietic stem cells.

Tumor cells can produce immune escape by various pathways, e.g.,down-regulating expression of the molecules that participate in T cellrecognition and antigen responses, or reducing immunogenicity, therebyenabling the immune system of an organism to be incapable of removingtumors effectively. Studies have found that chimeric antigen receptor Tcells (CAR-T cells) can recognize antigens on the surface of tumor cellsand specifically kill the tumor cells, and thus they are useful for thetreatment of tumors.

Lentiviruses prepared using a plasmid packaging system that arecurrently available in the prior art have a low concentration of viableviruses and a low transfection titer. To achieve an ideal effect of Tcell transfection, it is necessary to add a much higher dose oflentiviruses with the disadvantages of higher costs, excess residualsubstances and poor safety performance. In spite of the fact thatfour-plasmid packaging system has also been used in the prior art toreplace the three-plasmid packaging system, the proper ratio at whichthe plasmids can be combined to provide higher transfection titer whileensuring good safety performance has not been determined yet. Thus,there remains an urgent need to screen a suitable ratio of the fourplasmids for packaging lentiviral vectors to prepare modified immuneeffector cells.

SUMMARY OF THE INVENTION

To overcome the disadvantages of the prior art, the present disclosureprovides a four-plasmid packaging system composed of a target plasmidand three helper plasmids, wherein the target plasmid is freed fromnon-essential components for virus packaging, thereby effectivelyminimizing potential safety hazards. By using four plasmids atparticular ratios, the present disclosure can effectively increase thetransfection titer of the obtained lentivirus, and provide CAR-T cellsthat exhibit excellent clinical therapeutic effects and higher safetyperformance obtained by transfecting the T cells with lentiviral vectorsloaded with particular CAR.

The present disclosure provides a chimeric antigen receptor, comprisingan amino acid sequence shown in SEQ ID NO. 1. The present disclosurefurther provides a nucleic acid encoding the chimeric antigen receptor,a vector comprising the nucleic acid, an immune effector cell comprisingthe chimeric antigen receptor, the nucleic acid molecule and/or thevector, a method for preparing the immune effector cell, a compositioncomprising the immune effector cell, and use of the chimeric antigenreceptor.

The chimeric antigen receptor according to the present disclosure atleast comprises one of the following advantageous effects:

(1) stable expression on the surface of immune cells (such as T cells),at high expression levels;

(2) a strong ability to kill CD19-positive target cells;

(3) an ability to promote immune cells to secrete cytokines, e.g.,enhancing the ability of T cells to secrete cytokines (INF-γ or IL-6) byat least 1, 2, or 3 times;

(4) being non-hemolytic, and not susceptible to induce hemolysis oraggregation of red blood cells;

(5) being vascular irritation-free, and resulting in no local orsystemic abnormalities after administration;

(6) being devoid of oncogenic potential, and non-oncogenic in vivo or invitro;

(7) an ability to prolong the survival time of cancer patients;

(8) an ability to effectively ameliorate the severity of cancer (such asacute lymphoblastic leukemia in adults, acute lymphoblastic leukemia inchildren and/or non-Hodgkin's lymphoma); and (9) an ability to exhibithigher safety performance with a lower risk of inducing side effects(e.g., cytokine release syndrome or CAR-T-cell-related encephalopathysyndrome).

In one aspect, the present disclosure provides a plasmid combination,wherein the plasmid combination comprises plasmids Seq1, PMD2.G,pMDLg-pRRE and pRSV-Rev; and the plasmids Seq1, PMD2.G, pMDLg-pRRE andpRSV-Rev are present at a ratio of 2-6: 1-1.5: 1-3: 1-1.5.

In certain embodiments, the plasmid Seq1 in the plasmid combinationcomprises a nucleic acid molecule encoding the chimeric antigenreceptor, wherein the chimeric antigen receptor comprises an amino acidsequence shown in SEQ ID NO. 1.

In certain embodiments, the plasmid Seq1 in the plasmid combinationcomprises a nucleic acid molecule encoding the chimeric antigenreceptor, wherein the nucleic acid molecule comprises a nucleic acidsequence shown in SEQ ID NO. 2.

In certain embodiments, the plasmids Seq1, PMD2.G, pMDLg-pRRE andpRSV-Rev in the plasmid combination are present at a ratio of 11.8:3.53: 6.33: 2.3, 13.8: 3.48: 5.31: 2.54 or 14: 4.67: 4.67: 4.67.

In one aspect, the present disclosure provides a method for preparing alentiviral vector, the method comprising the step of introducing theplasmid combination into a cell. In certain embodiments, the step ofintroducing refers to the step of transfecting.

In one aspect, the present disclosure provides a method for obtaining alentiviral vector by transfecting cells with a four-plasmid packagingsystem, wherein the plasmid packaging system comprises plasmids Seq1,PMD2.G, pMDLg-pRRE and pRSV-Rev, and the plasmids Seq1, PMD2.G,pMDLg-pRRE and pRSV-Rev are present at a ratio of 2-6: 1-1.5: 1-3:1-1.5.

In certain embodiments, the plasmid Seq1 can express a chimeric antigenreceptor, comprising an amino acid sequence shown in SEQ ID NO.1.

In certain embodiments, the plasmid Seq1 comprises an isolated nucleicacid molecule encoding a chimeric antigen receptor, wherein the nucleicacid molecule comprises a nucleic acid sequence shown in SEQ ID NO.2.

In certain embodiments, the plasmids Seq1, PMD2.G, pMDLg-pRRE andpRSV-Rev are present ata ratio of 11.8: 3.53: 6.33: 2.3 or 13.8: 3.48:5.31: 2.54.

In certain embodiments, the cell is 293T. In certain embodiments, thecell is 293T/17.

In one aspect, the present disclosure provides a chimeric antigenreceptor, comprising an amino acid sequence shown in SEQ ID NO. 1.

In another aspect, the present disclosure further provides an isolatednucleic acid molecule encoding the chimeric antigen receptor describedherein.

In another aspect, the present disclosure further provides an isolatednucleic acid molecule encoding a chimeric antigen receptor, wherein thenucleic acid molecule comprises a nucleic acid sequence shown in SEQ IDNO. 2.

In another aspect, the present disclosure further provides a plasmidcomprising the nucleic acid molecule described herein.

In another aspect, the present disclosure further provides a method forpreparing a modified immune effector cell, comprising the step ofpreparing and obtaining the lentiviral vector according to the methodfor preparing a lentivirus.

In certain embodiments, the method further comprises the step ofintroducing the lentiviral vector into an immune effector cell. Incertain embodiments, the step of introducing refers to the step oftransfecting.

In certain embodiments, the immune effector cell is selected from thegroup consisting of a T lymphocyte and a natural killer cell.

In another aspect, the present disclosure further provides a modifiedimmune effector cell comprising the modified immune effector cellprepared and obtained by the method for preparing the modified immuneeffector cell.

In certain embodiments, the immune effector cells are selected from thegroup consisting of T lymphocytes and natural killer cells.

In certain embodiments, the immune effector cells can express thechimeric antigen receptor described herein.

In another aspect, the present disclosure further provides a compositioncomprising the modified immune effector cells obtained by the methoddescribed herein.

In certain embodiments, the immune effector cell is selected from thegroup consisting of T lymphocytes and natural killer cells.

In certain embodiments, the chimeric antigen receptor described hereinis expressed on the surface of the immune effector cell.

In another aspect, the present disclosure further provides use of theimmune effector cell prepared by the method, and/or the compositioncomprising the immune effector cell, the chimeric antigen receptor, thenucleic acid molecule, the vector and/or the immune effector cell in themanufacture of a medicament, wherein the medicament is useful for thetreatment of a disease or disorder associated with CD19 expression.

In another aspect, the present disclosure further provides a method forthe treatment of a disease or disorder associated with CD19 expression,comprising applying the composition and/or the immune effector cell.

In another aspect, the present disclosure further provides thecomposition and/or the immune effector cell for use in the treatment ofa disease or disorder associated with CD19 expression.

In certain embodiments, the disease or disorder associated with CD19expression comprises non-solid tumors.

In certain embodiments, the non-solid tumor comprises leukemia and/orlymphoma.

In certain embodiments, the disease or disorder associated with CD19expression comprises acute lymphoblastic leukemia and/or B-celllymphoma.

In certain embodiments, the acute lymphoblastic leukemia comprises acutelymphoblastic leukemia in adults and/or acute lymphoblastic leukemia inchildren.

In certain embodiments, the medicament for treating acute lymphoblasticleukemia is administered at a dose of 0.25×10⁸ to 0.5×10⁸ CAR-positive Tcells.

In certain embodiments, the B-cell lymphoma comprises non-Hodgkin'slymphoma.

In certain embodiments, the medicament for treating non-Hodgkin'slymphoma is administered at a dose of 1×10⁸ to 2×10⁸ CAR-positive Tcells.

Persons skilled in the art can easily perceive other aspects andadvantages of the present disclosure from the detailed descriptionbelow. In the following detailed description, only exemplary embodimentsof the present disclosure are shown and described. As persons skilled inthe art will recognize, the contents of this disclosure enable personsskilled in the art to make modifications to the specific embodimentsdisclosed herein without departing from the inventive spirit and scopeof this disclosure. Correspondingly, the drawings and illustrations inthe description of the present disclosure are only exemplary, ratherthan restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The specific features involved in this disclosure are shown in theappended claims. The characteristics and advantages of the disclosureinvolved herein can be better understood by referring to the exemplaryembodiments and the accompanying drawings described in detail below. Abrief description of the drawings is as follows:

FIG. 1A shows the transfection titer after the transfection with thefour-plasmid system.

FIG. 1B shows the physical titer after the transfection with thefour-plasmid system.

FIG. 2A shows the detection results of CAR molecules expressed on thesurface of CNCT19 cells.

FIG. 2B shows the detection results of CAR molecules expressed on thesurface of CNCT19 cells.

FIG. 3 shows residual rates of tumor cells under different co-cultureconditions.

FIG. 4 shows killing of target cells (CHO-CD19) by CNCT19 cells asmonitored in real time by the real-time cell analysis (RTCA) dualpurpose (DP) system.

FIG. 5A shows variations of INF-γ concentrations in the supernatantunder different co-culture conditions.

FIG. 5B shows variations of IL-6 concentrations in the supernatant underdifferent co-culture conditions.

FIG. 6A shows observation results of each test tube before shaking for 3hours.

FIG. 6B shows observation results of each test tube after shaking for 3hours.

FIG. 7A shows a micrograph of the locally injected site afteradministration of the CAR-T cells (HE staining, 10×objective lens).

FIG. 7B shows a micrograph of the locally injected site afteradministration of sodium chloride injection solution (HE staining,10×objective lens).

FIG. 8 shows soft-agar colony formation after 3 weeks of cellinoculation in each group.

FIG. 9 shows survival curves of NCG mice with Nalm-6 xenograft tumorstreated with different cells.

FIG. 10 shows distribution of tissues after single administration ofCNCT19 cells.

FIG. 11 is a comparison of the CNCT19 cells distributed in vivo intumor-bearing and non-tumor-bearing animals.

FIG. 12 shows variations in the CNCT19 cells distributed in varioustissues of the animals after single administration.

DETAILED DESCRIPTION OF THE INVENTION

The following specific examples illustrate the particular embodiments ofthe disclosure. Persons familiar with this technology can easilyunderstand the other advantages and effects of the disclosure from thecontents disclosed herein.

Hereinafter, the disclosure is further described: according to thepresent disclosure, unless otherwise specified, the scientific andtechnical terms used herein have the meanings commonly understood bythose skilled in the art. In addition, the related terms and laboratoryprocedures of protein and nucleic acid chemistry, molecular biology,cell and tissue culture, microbiology, and immunology used herein areall terms and routine procedures widely used in the correspondingfields. In the meantime, to better understand the present disclosure,definitions and explanations of the related terms are provided below.

As used herein, the term “Chimeric Antigen Receptor” (CAR) generallyrefers to a fused protein comprising an extracellular domain capable ofbinding to an antigen and at least one intracellular domain. CAR is acore component of chimeric antigen receptor T cells (CAR-T), which mayinclude a targeting moiety (for example, a moiety binding atumor-associated antigen (TAA)), a hinge region, a transmembrane region,and an intracellular domain. In the present disclosure, the CAR may becombined with the intracellular domain for T cell receptor activationbased on the antigen specificity of an antibody. T cells expressing CARcan specifically recognize and eliminate malignant cells expressing thetarget antigen.

As used herein, the term “isolated” generally refers to being obtainedby artificial means from the natural state. If certain “isolated”substance or component appears in nature, it might mean that either thenatural environment in which it is located has been changed, or thesubstance has been isolated from the natural environment, or both. Forexample, certain non-isolated polynucleotide or polypeptide naturallyoccurs in a living animal, and the same polynucleotide or polypeptidehaving high purity and isolated from this natural state is called as theisolated one. The term “isolated” does not exclude the occasion of beingmixed with an artificial or synthetic substance, nor exclude thepresence of other impurities that do not impair the activity of the substance.

As used herein, the term “immune effector cell” generally refers tocells that participate in an immune response, such as those promoting animmune effector response. In the present disclosure, the immune effectorcell may be selected from the group consisting of T lymphocytes andnatural killer cells.

As used herein, the term “specifically binds and/or specificallyrecognizes” generally refers to an interaction that is measurable andreproducible, such as the binding between a target and an antibody (orCAR structural fragment), which may determine the presence of a targetwhen a heterogeneous cell population of a molecule (including abiomolecule) exists. For example, an antibody (or CAR structuralfragment) that specifically binds to a target (which may be an epitope)is the antibody (or CAR structural fragment) that binds to the targetwith higher affinity and avidity, in an easier manner and/or for alonger duration, compared with its binding to other targets.

As used herein, the term “isolated nucleic acid molecule” generallyrefers to a nucleotide, deoxyribonucleotide or ribonucleotide of anylength in its isolated form, or an analog that has been isolated fromtheir natural environment or artificially synthesized.

As used herein, the term “plasmid” or “vector” generally refers to atool for delivering nucleic acid into which a polynucleotide encodingcertain protein can be inserted and by which the protein can beexpressed. The vector can be transformed, transduced or transfected intothe host cell so that the genetic material element it carries can beexpressed in the host cell. For example, vectors include a plasmid; aphagemid; a cosmid; an artificial chromosome (such as yeast artificialchromosome (YAC), a bacterial artificial chromosome (BAC) or aP1-derived artificial chromosome (PAC)); a phage such as X, phage or M13phage and an animal virus, etc. The types of animal viruses as thevector include retrovirus (including lentivirus), adenovirus,adeno-associated virus, herpes virus (such as herpes simplex virus),poxvirus, baculovirus, papilloma virus, and papilloma vacuole virus(such as SV40). A vector may contain a variety of elements that controlexpression, including promoter sequences, transcription initiationsequences, enhancer sequences, selective elements, and reporter genes.In addition, the vector may also contain an origin of replication. Thevector may also include components that help its entry into the cells,such as viral particles, liposomes or protein coats, but are not limitedto those substances.

As used herein, the term “composition” generally refers to a compositionsuitable for administration to a patient. For example, the compositionaccording to the present disclosure may comprise the immune effectorcells described herein. Furthermore, the composition may also compriseone or more suitable formulations of (pharmaceutically effective)carriers, stabilizers, excipients, diluents, solubilizers, surfactants,emulsifiers and/or preservatives. Acceptable ingredients of thecomposition are non-toxic to the recipient at any dose and concentrationused. The compositions of the present disclosure include, but are notlimited to, liquids, and frozen or lyophilized compositions.

As used herein, the term “CD19” usually refers to cluster ofdifferentiation (CD) 19 proteins, which is the cluster of antigenicdeterminants that can be detected on leukemia precursor cells. The aminoacid and nucleic acid sequences of human and murine CD19 can be found inpublic databases (such as GenBank, UniProt, and Swiss-Prot). Forexample, the amino acid sequence of human CD19 can be accessed underUniProt/Swiss-Prot Accession Number P15391, and the nucleotide sequenceencoding human CD19 can be accessed under Accession Number NM_001178098.According to the present disclosure, “CD19” may include proteins withmutations (for example, point mutations, fragments, insertions,deletions, and splice variants of full-length wild-type CD19).

As used herein, the term “subject” generally refers to a human ornon-human animal, including but not limited to cat, dog, horse, pig,cow, sheep, rabbit, mouse, rat, or monkey.

As used herein, the term “lentiviral vector” generally refers to avector comprising one or more nucleic acid sequences derived from atleast part of the lentiviral genome. The lentiviral vector may comprisenon-coding sequences of one or more proteins from the lentivirus.

As used herein, the term “target plasmid” may include, for example, aheterologous nucleic acid sequence (for example, a nucleic acid sequenceencoding a CAR) to be transferred into a cell, and may further include,for example, one or more lentiviral genes or parts thereof.

As used herein, “helper plasmid” may contain one or more genes encodinglentiviral proteins or parts thereof. For example, a gene encoding alentiviral capsid protein may be included, and for another example, agene encoding an env protein or parts thereof may be included. The hostcell can be transfected with a target plasmid and one or more helperplasmids to produce a virus, which can be used to infect a target cell(for example, a T cell) to express in the target cell one or moretransgenes (for example, a gene encoding CAR) contained in theheterologous nucleic acid sequence.

As used herein, the term “comprising” generally refers to the inclusionof explicitly specified features, but not excluding other elements.

As used herein, the term “about” generally refers to vary within a rangeof 0.5%-10% greater or less than the stated value, such as varyingwithin a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%,5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% greater or less thanthe stated value.

Four-Plasmid System

In one aspect, the present disclosure provides a plasmid combination,wherein the plasmid combination comprises plasmids Seq1, PMD2.G,pMDLg-pRRE and pRSV-Rev.

A four-plasmid system is used in the present disclosure to transfectlentivirus according to the method described in its instructions. Thefour-plasmid system may include a target plasmid (Seq1) and three helperplasmids (PMD2.G, pMDLg-pRRE and pRSV-Rev).

According to the present disclosure, the target plasmid may be Seq1. TheSeq 1 carries the gene sequence of interest. The gene of interest in thevector plasmid is inserted into the genome of the target cell (293 cell)by the lentiviral vector to achieve stable expression of CAR. Thetransfer plasmid has an amino acid sequence shown in SEQ ID NO.1, and anucleic acid sequence shown in SEQ ID NO.2.

Helper plasmid 1 may be PMD2.G, which is used to provide a VSV-G geneencoding vesicular stomatitis virus glycoprotein G. TheVSVG-envelope-pseudotyped lentiviral vector extends the target celltropism range of the vector and increases the stability of thelentiviral vector, thereby allowing the lentiviral vector to beconcentrated by high-speed centrifugation, and further resulting inhigher titer.

Helper plasmid 2 may be pMDLg-pRRE, which is used to provide a Revprotein-binding site and contains Gag and Pol genes. The Gag geneencodes major structural proteins of viral particles, such asnucleocapsid protein, membrane protein and capsid protein. The pol geneencodes viral replication-associated enzymes, such as protease, reversetranscriptase and integrase. It plays a vital role in virus assembly.

Helper plasmid 3 may be pRSV-Rev, which is used to provide a Rev genefor regulating the expression levels of the Gag and Pol genes andguiding the replication process of the single-stranded DNA, and canregulate splicing/RNA transport. It plays an important role in virusassembly.

In certain embodiments, the plasmids Seq1, PMD2.G, pMDLg-pRRE andpRSV-Rev may be present in the plasmid combination ata ratio of 2-6:1-1.5: 1-3: 1-1.5, such as 2-5: 1-1.5: 1-3: 1-1.5, 2-4: 1-1.5: 1-3:1-1.5, 2-3: 1-1.5:1-3: 1-1.5, 3-6: 1-1.5: 1-3: 1-1.5, 3-5: 1-1.5: 1-3:1-1.5, 3-4: 1-1.5: 1-3: 1-1.5, 4-6: 1-1.5: 1-3: 1-1.5, 4-5: 1-1.5: 1-3:1-1.5, or 5-6: 1-1.5: 1-3: 1-1.5. In certain embodiments, the plasmidsSeq1, PMD2.G, pMDLg-pRRE and pRSV-Rev may be present at a ratio of 2-6:1-1.5: 1-3: 1-1.5, such as 2.1: 1-1.5: 1-3: 1-1.5, 2.2: 1-1.5: 1-3:1-1.5, 2.3: 1-1.5: 1-3: 1-1.5, 2.4: 1-1.5: 1-3: 1-1.5, 2.5: 1-1.5: 1-3:1-1.5, 2.6: 1-1.5: 1-3: 1-1.5, 2.7: 1-1.5: 1-3: 1-1.5, 2.8: 1-1.5: 1-3:1-1.5, 2.9: 1-1.5: 1-3: 1-1.5, 3.0: 1-1.5: 1-3: 1-1.5, 3.1: 1-1.5: 1-3:1-1.5, 3.2: 1-1.5: 1-3: 1-1.5, 3.3: 1-1.5: 1-3: 1-1.5, 3.4: 1-1.5: 1-3:1-1.5, 3.5: 1-1.5: 1-3: 1-1.5, 3.6: 1-1.5: 1-3: 1-1.5, 3.7: 1-1.5: 1-3:1-1.5, 3.8: 1-1.5: 1-3: 1-1.5, 3.9: 1-1.5: 1-3: 1-1.5, 4.0: 1-1.5: 1-3:1-1.5, 4.1: 1-1.5: 1-3: 1-1.5, 4.2: 1-1.5: 1-3: 1-1.5, 4.3: 1-1.5: 1-3:1-1.5, 4.4: 1-1.5: 1-3: 1-1.5, 4.5: 1-1.5: 1-3: 1-1.5, 4.6: 1-1.5: 1-3:1-1.5, 4.7: 1-1.5: 1-3: 1-1.5, 4.8: 1-1.5: 1-3: 1-1.5, 4.9: 1-1.5: 1-3:1-1.5, 5.0: 1-1.5: 1-3: 1-1.5, 5.1: 1-1.5: 1-3: 1-1.5, 5.2: 1-1.5: 1-3:1-1.5, 5.3: 1-1.5: 1-3: 1-1.5, 5.4: 1-1.5: 1-3: 1-1.5, 5.5: 1-1.5: 1-3:1-1.5, 5.6: 1-1.5: 1-3: 1-1.5, 5.7: 1-1.5: 1-3:1-1.5, 5.8: 1-1.5: 1-3:1-1.5, 5.9: 1-1.5: 1-3: 1-1.5, or 6.0: 1-1.5: 1-3: 1-1.5.

In certain embodiments, the plasmids Seq1, PMD2.G, pMDLg-pRRE andpRSV-Rev may be present in the plasmid combination ata ratio of 2-6:1-1.5: 1-3:1-1.5, such as 2-6: 1-1.3: 1-3: 1-1.5, 2-6: 1.2-1.5: 1-3:1-1.5, or 2-6: 1.1-1.4: 1-3: 1-1.5. In certain embodiments, the plasmidsSeq1, PMD2.G, pMDLg-pRRE and pRSV-Rev may be present at a ratio of 2-6:1-1.5: 1-3: 1-1.5, such as 2-6: 1.1: 1-3: 1-1.5, 2-6: 1.2: 1-3: 1-1.5,2-6: 1.3: 1-3: 1-1.5, 2-6: 1.4: 1-3: 1-1.5, or 2-6: 1.5: 1-3: 1-1.5.

In certain embodiments, the plasmids Seq1, PMD2.G, pMDLg-pRRE andpRSV-Rev may be present in the plasmid combination ata ratio of 2-6:1-1.5: 1-3: 1-1.5, such as 2-6: 1-1.5: 1-2.5: 1-1.5, 2-6: 1-1.5: 1-2.0:1-1.5, 2-6: 1-1.5: 1-1.5: 1-1.5, 2-6: 1-1.5: 1.5-3: 1-1.5, 2-6: 1-1.5:1.5-2.5: 1-1.5, 2-6: 1-1.5: 1.5-2: 1-1.5, 2-6: 1-1.5: 2-3: 1-1.5, 2-6:1-1.5: 2-2.5: 1-1.5, or 2-6: 1-1.5: 2.5-3: 1-1.5. In certainembodiments, the plasmids Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev may bepresent ata ratio of 2-6: 1-1.5: 1-3: 1-1.5, such as 2-6: 1-1.5: 1.1:1-1.5, 2-6: 1-1.5: 1.2: 1-1.5, 2-6: 1-1.5: 1.3: 1-1.5, 2-6: 1-1.5: 1.4:1-1.5, 2-6: 1-1.5: 1.5: 1-1.5, 2-6: 1-1.5: 1.6: 1-1.5, 2-6: 1-1.5: 1.7:1-1.5, 2-6: 1-1.5: 1.8: 1-1.5, 2-6: 1-1.5: 1.9: 1-1.5, 2-6: 1-1.5: 2:1-1.5, 2-6: 1-1.5: 2.1: 1-1.5, 2-6: 1-1.5: 2.2: 1-1.5, 2-6: 1-1.5: 2.3:1-1.5, 2-6: 1-1.5: 2.4: 1-1.5, 2-6: 1-1.5: 2.5: 1-1.5, 2-6: 1-1.5: 2.6:1-1.5, 2-6: 1-1.5: 2.7: 1-1.5, 2-6: 1-1.5: 2.8: 1-1.5, 2-6: 1-1.5: 2.9:1-1.5, or 2-6: 1-1.5: 3: 1-1.5.

In certain embodiments, the plasmids Seq1, PMD2.G, pMDLg-pRRE andpRSV-Rev may be present in the plasmid combination at a ratio of 11.8:3.53: 6.33: 2.3, 13.8: 3.48: 5.31: 2.54 or 14: 4.67: 4.67: 4.67. Incertain embodiments, the plasmids Seq1, PMD2.G, pMDLg-pRRE and pRSV-Revmay be present at a ratio of 11.8: 3.53: 6.33: 2.3. In certainembodiments, the plasmids Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev may bepresent at a ratio of 13.8: 3.48: 5.31: 2.54. In certain embodiments,the plasmids Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev may be present at aratio of 14: 4.67: 4.67: 4.67.

Chimeric Antigen Receptor, Nucleic Acid, Vector, Immune Effector Cell,and Composition

The plasmid Seq1 of the present disclosure can express a chimericantigen receptor, comprising the amino acid sequence shown in SEQ IDNO.1. The present disclosure further provides a chimeric antigenreceptor, comprising an amino acid sequence having at least 80% (such asat least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%) sequence identity with the amino acidsequence shown in SEQ ID NO. 1.

In certain embodiments, the chimeric antigen receptor described hereincan specifically bind to and/or recognize tumor antigens. For example,the chimeric antigen receptor described herein can specifically bind toand/or recognize CD19 antigen.

In certain embodiments, the chimeric antigen receptor described hereincan promote an immune effector cell to secrete cytokine. The immuneeffector cell may be selected from the group consisting of T lymphocytesand natural killer cells. The cytokine may be selected from the groupconsisting of IFN-γ and IL-6. The immune effector cell may be amammalian immune effector cell. The T lymphocyte may be a mammalian Tlymphocyte, and the natural killer cell may also be a mammalian naturalkiller cell. The T lymphocyte may be a human T lymphocyte, and thenatural killer cell may also be a human natural killer cell.

In certain embodiments, the chimeric antigen receptor described hereinis non-hemolytic and vascular irritation-free.

In certain embodiments, the chimeric antigen receptor described hereinis non-oncogenic in vitro.

In certain embodiments, the chimeric antigen receptor described hereinis non-oncogenic in vivo.

In certain embodiments, the chimeric antigen receptor described hereincan effectively treat tumors. The tumor may be a CD19-positive tumor.For example, the chimeric antigen receptor described herein caneffectively prolong the survival time of patients with CD19-positivetumors. For example, the chimeric antigen receptor described herein caneffectively prolong the survival time of patients with non-solid tumors.For example, the chimeric antigen receptor described herein caneffectively prolong the survival time of lymphoma and/or leukemia. Foranother example, the chimeric antigen receptor described herein caneffectively prolong the survival time of adult patients with acutelymphoblastic leukemia. For another example, the chimeric antigenreceptor described herein can effectively prolong the survival time ofchildren patients with acute lymphoblastic leukemia. For anotherexample, the chimeric antigen receptor described herein can effectivelyprolong the survival time of patients with B-cell lymphoma (for example,non-Hodgkin's lymphoma).

In certain embodiments, the chimeric antigen receptor described hereincan effectively treat acute lymphoblastic leukemia in adults.

In certain embodiments, the chimeric antigen receptor described hereincan effectively treat acute lymphoblastic leukemia in children.

In certain embodiments, the chimeric antigen receptor described hereincan effectively treat non-Hodgkin's lymphoma.

The plasmid Seq1 of the present disclosure comprises an isolated nucleicacid molecule encoding the chimeric antigen receptor described herein.

The plasmid Seq1 of the present disclosure comprises an isolated nucleicacid molecule encoding a chimeric antigen receptor, wherein the nucleicacid molecule comprises the nucleic acid sequence shown in SEQ ID NO.2.

The plasmid Seq1 of the present disclosure comprises an isolated nucleicacid molecule encoding a chimeric antigen receptor, wherein the nucleicacid molecule comprises a nucleic acid sequence analogous to thesequence shown in SEQ ID NO.2 and is a nucleic acid molecule encodingthe chimeric antigen receptor.

In certain embodiments, the nucleic acid sequence analogous to thesequence shown in SEQ ID NO.2 refers to a nucleic acid sequence havingat least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequenceidentity with the nucleic acid sequence shown in SEQ ID NO. 2.

In certain embodiments, the nucleic acid sequence analogous to thesequence shown in SEQ ID NO.2 means that the nucleic acid molecule canencode the chimeric antigen receptor, though it is different from thenucleic acid sequence shown in SEQ ID NO. 2 owing to the wobble(degeneracy) of the base at position 3 of the nucleic acid codon.

The present disclosure includes variants of genes and proteins (forexample, variants of the amino acid sequence shown in SEQ ID NO.1, orvariants of the nucleic acid sequence shown in SEQ ID NO. 2 as describedherein), which retain one or more biological activities. Such variantsof the protein or polypeptide include a protein or polypeptide that hasbeen or can be modified using recombinant DNA technology so that theprotein or polypeptide has altered or additional properties; forexample, the variant confers enhanced stability in plasma or increasedactivity to the protein. The variant may be different from the referencesequence, e.g., being different from a naturally occurringpolynucleotide, protein or peptide. At the nucleotide sequence level,the naturally occurring variant gene and the non-naturally occurringvariant gene will typically have at least about 50%, more typically atleast about 70%, and even more typically at least about 80% identity(90% or higher identity) to the reference gene. At the amino acidsequence level, the naturally occurring variant protein and thenon-naturally occurring variant protein will typically have at leastabout 70%, more typically at least about 80%, and even more typically atleast about 90% or higher identity to the reference protein, whileallowing higher percent non-identity regions in non-conserved regions(e.g., the percent identity being less than 70%, such as less than 60%,less than 50%, or even less than 40%). In other embodiments, thesequence has at least 60%, 70%, 75% or more identity (e.g., 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or higher identity) with the referencesequence. The modification procedures for introducing a nucleotide andan amino acid into a polynucleotide, protein, or polypeptide have beenknown to those skilled in the art (see, for example, Sambrook et al.(1989)).

As used herein, the term “identity”, “homology” and their grammaticalvariants generally mean that two or more entities are identical whentheir sequences are “aligned”. Thus, for example, when two polypeptideshave identical sequences, they have the same amino acid sequence atleast within the reference regions or parts. If two polynucleotides haveidentical sequences, they have the same polynucleotide sequence at leastwithin the reference regions or parts. The identity can be the identityof the defined zones (regions or domains) of the sequences. The “zones”or “regions” of identity refer to the same parts of two or morereference entities. Therefore, if two proteins or nucleic acid sequencesare the same in one or more sequence zones or regions, they haveidentity in that region. “Aligned” sequences refer to morepolynucleotide or protein (amino acid) sequences, which often containsupplementary or additional bases or amino acids (gaps) compared withthe reference sequence. The degree of identity (homology) between twosequences can be determined using computer programs and mathematicalalgorithms. Such algorithms that calculate percent sequence identity(homology) generally calculate sequence gaps and mismatches in thecompared regions or zones. For example, BLAST (for example, BLAST 2.0)search algorithm (see, for example, Altschul et al., J.Mol. Biol. 215:403 (1990), publicly available from NCBI) gives exemplary searchparameters as follows: Mismatch −2, gap opening 5, gap extension 2.

According to the present disclosure, the nucleic acid molecule may be anucleotide, deoxyribonucleotide or ribonucleotide of any length in itsisolated form, or an analog that has been isolated from their naturalenvironment or artificially synthesized, as long as it is capable ofencoding the chimeric antigen receptor described herein.

In another aspect, the present disclosure provides a vector comprisingthe nucleic acid molecule described herein.

According to the present disclosure, the vector can be transformed,transduced or transfected into the host cell so that the geneticmaterial element it carries can be expressed in the host cell. Forexample, vectors include a plasmid; a phagemid; a cosmid; an artificialchromosome (such as yeast artificial chromosome (YAC), a bacterialartificial chromosome (BAC) or a P1-derived artificial chromosome(PAC)); a phage such as X, phage or M13 phage and an animal virus, etc.The types of animal viruses as the vector include retrovirus (includinglentivirus), adenovirus, adeno-associated virus, herpes virus (such asherpes simplex virus), poxvirus, baculovirus, papilloma virus, andpapilloma vacuole virus (such as SV40). For another example, the vectormay contain a variety of elements that control expression, includingpromoter sequences, transcription initiation sequences, enhancersequences, selective elements, and reporter genes. In addition, thevector may also contain an origin of replication. Moreover, the vectormay also include components that help its entry into the cells, such asviral particles, liposomes or protein coats, but are not limited tothose substances.

In one aspect, the present disclosure provides a method for preparing alentiviral vector, the method comprising introducing the plasmidcombination into a cell.

In another aspect, the present disclosure provides an immune effectorcell, comprising the chimeric antigen receptor, the nucleic acidmolecule and/or the vector described herein.

According to the present disclosure, the immune effector cell may beselected from the group consisting of T lymphocytes and natural killercells. In certain embodiments, the immune effector cell may be a humanimmune effector cell. For example, the immune effector cell can be ahuman T lymphocyte. For another example, the immune effector cell can bea human natural killer cell.

According to the present disclosure, the chimeric antigen receptordescribed herein is expressed on the surface of the immune effectorcell.

In certain embodiments, the immune effector cell described herein caneffectively kill tumor cells. The tumor cells may be CD19-positivecells. For example, the immune effector cell described herein canconsiderably lower the residual rate of CD19-positive human leukemiacell line Nalm-6 cells.

In certain embodiments, the immune effector cell described herein caneffectively promote cytokine secretion when it comes into contact withCD19-positive cells. The cytokine may be selected from the groupconsisting of IFN-γ and IL-6. For example, co-culturing the immuneeffector cell described herein with CD19-positive human leukemia cellline Nalm-6 cells results in a significant increase in the secretion ofIFN-γ and IL-6 cytokine.

In certain embodiments, the immune effector cell described herein isnon-hemolytic and vascular irritation-free. For example, in hemolysistest in vitro, the immune effector cell described herein would notinduce hemolysis and blood aggregation. For another example, the immuneeffector cell described herein is vascular irritation-free.

In certain embodiments, the immune effector cell described herein isnon-oncogenic in vitro.

In certain embodiments, the immune effector cell described herein isnon-oncogenic in vivo.

In certain embodiments, the immune effector cell described herein caneffectively treat tumors. The tumor may be a CD19-positive tumor. Forexample, the immune effector cell described herein can effectivelyprolong the survival time of patients with CD19-positive tumors. Foranother example, the immune effector cell described herein caneffectively prolong the survival time of adult patients with acutelymphoblastic leukemia. For another example, the immune effector celldescribed herein can effectively prolong the survival time of childrenpatients with acute lymphoblastic leukemia. For another example, theimmune effector cell described herein can effectively prolong thesurvival time of patients with B-cell lymphoma (such as non-Hodgkin'slymphoma).

In certain embodiments, the immune effector cell described herein caneffectively treat acute lymphoblastic leukemia in adults.

In certain embodiments, the immune effector cell described herein caneffectively treat acute lymphoblastic leukemia in children.

In certain embodiments, the immune effector cell described herein caneffectively treat B-cell lymphoma (such as non-Hodgkin's lymphoma).

In another aspect, the present disclosure provides a compositioncomprising the immune effector cell described herein.

According to the present disclosure, the composition may also compriseone or more suitable formulations of (pharmaceutically effective)carriers, stabilizers, excipients, diluents, solubilizers, surfactants,emulsifiers and/or preservatives. Acceptable ingredients of thecomposition are non-toxic to the recipient at any dose and concentrationused. The compositions of the present disclosure include, but are notlimited to, liquids, and frozen or lyophilized compositions.

In certain embodiments, the composition may be a composition forparenteral, transdermal, intraluminal, intraarterial, intrathecal and/orintranasal administration or by direct injection into tissue. Forexample, the composition may be administered to a patient or subject viainfusion or injection. In other embodiments, administration of thecomposition may be effected by different ways, e.g., by intravenous,intraperitoneal, subcutaneous, intramuscular, topical or intradermaladministration. In other embodiments, the composition can beadministered uninterruptedly. The uninterrupted (or continuous)administration may be realized by a small pump system worn by thepatient for metering the influx of therapeutic agent into the body ofthe patient, as described in WO2015/036583.

According to the present disclosure, the dosage regimen of thecomposition may be a dose of a rapid infusion agent, multiple divideddoses administered over time, or the doses may be decreased or increasedin proportion to the severity and urgency of the treatment situation. Incertain embodiments, the treatment regimen can be administered once aweek, once every two weeks, once every three weeks, once every fourweeks, once a month, once every three months, or once every three to sixmonths. In certain embodiments, the dosage regimen includes intravenousadministration, and the dose may be administered in a range of 0.1×10⁸to 3×10⁸ CAR-positive T cells, for example, 0.15×10⁸ to 2×10⁸CAR-positive T cells, 0.5×10⁸ to 2×10⁸ CAR-positive T cells, 1×10⁸ to2×10⁸ CAR-positive T cells, 0.2×10⁸ to 2×10⁸ CAR-positive T cells,0.2×10⁸ to 1×10⁸ CAR-positive T cells, 0.25×10⁸ to 1×10⁸ CAR-positive Tcells, 0.25×10⁸ to 0.5×10⁸ CAR-positive T cells, or 0.5×10⁸ CAR-positiveT cells, or 2×10⁸ CAR-positive T cells.

The doses for administration may vary for different indications. Incertain embodiments, the immune effector cell for the treatment of adultpatients with relapsed and refractory acute lymphoblastic leukemia maybe administered at a dose of 0.25×10⁸ to 0.5×10⁸ CAR-positive T cells,or 0.5×10⁸ CAR-positive T cells, e.g., 0.3×10⁸ to 0.5×10⁸, 0.4×10⁸ to0.5×10⁸, 0.25×10⁸ to 0.4×10⁸, 0.3×10⁸ to 0.4×10⁸, or 0.4×10⁸ to 0.5×10⁸CAR-positive T cells. In certain embodiments, the immune effector cellfor the treatment of adult patients with relapsed and refractory acutelymphoblastic leukemia may be administered at a dose of 0.25×10⁸,0.26×10⁸, 0.27×10⁸, 0.28×10⁸, 0.29×10⁸, 0.3×10⁸, 0.31×10⁸, 0.32×10⁸,0.33×10⁸, 0.34×10⁸, 0.35×10⁸, 0.36×10⁸, 0.37×10⁸, 0.38×10⁸, 0.39×10⁸,0.4×10⁸, 0.41×10⁸, 0.42×10⁸, 0.43×10⁸, 0.44×10⁸, 0.45×10⁸, 0.46×10⁸,0.47×10⁸, 0.48×10⁸, 0.49×10⁸ or 0.5×10⁸ CAR-positive T cells.

In certain embodiments, the immune effector cell for the treatment ofchildren patients with relapsed and refractory acute lymphoblasticleukemia may be administered at a dose of 0.25×10⁸ to 0.5×10⁸CAR-positive T cells, or 0.5×10⁸ CAR-positive T cells, e.g., 0.3×10⁸ to0.5×10⁸, 0.4×10⁸ to 0.5×10⁸, 0.25×10⁸ to 0.4×10⁸, 0.3×10⁸ to 0.4×10⁸, or0.4×10⁸ to 0.5×10⁸ CAR-positive T cells. In some embodiments, the immuneeffector cell for the treatment of children patients with relapsed andrefractory acute lymphoblastic leukemia may be administered at a dose of0.25×10⁸, 0.26×10⁸, 0.27×10⁸, 0.28×10⁸, 0.29×10⁸, 0.3×10⁸, 0.31×10⁸,0.32×10⁸, 0.33×10⁸, 0.34×10⁸, 0.35×10⁸, 0.36×10⁸, 0.37×10⁸, 0.38×10⁸,0.39×10⁸, 0.4×10⁸, 0.41×10⁸, 0.42×10⁸ , 0.43×10⁸, 0.44×10⁸, 0.45×10⁸,0.46×10⁸, 0.47×10⁸, 0.48×10⁸, 0.49×10⁸ or 0.5×10⁸ CAR-positive T cells.

In other embodiments, the immune effector cell for the treatment ofpatients with relapsed and refractory non-Hodgkin's lymphoma may beadministered at a dose of 1×10⁸ to 2×10⁸ CAR-positive T cells, or 2×10⁸CAR-positive T cells, e.g., 1×10⁸ to 1.8×10⁸, 1×10⁸ to 1.5×10⁸, 1×10⁸ to1.3×10⁸, 1.3×10⁸ to 2×10⁸, 1.3×10⁸ to 1.5×10⁸, 1.5×10⁸ to 2×10⁸, 1.5×10⁸to 1.8×10⁸ or 1.8×10⁸ to 2×10⁸ CAR-positive T cells. In otherembodiments, the immune effector cell for the treatment of patients withrelapsed and refractory Hodgkin's lymphoma may be administered at a doseof 1×10⁸ 1.1×10⁸ 1.2×10⁸ 1.3×10⁸ 1.4×10⁸ 1.5×10⁸ 1.6×10⁸ 1.7×10⁸1.8×10⁸, 1.9×10⁸ or 2.0×10⁸ CAR-positive T cells.

Preparation Method and Use

In another aspect, the present disclosure further provides a method forpreparing an immune effector cell, comprising a step of transducing theplasmid or vector described herein into the immune effector cell.

In certain embodiments, the immune effector cell is selected from thegroup consisting of T lymphocytes and natural killer cells.

In another aspect, the present disclosure further provides use of thechimeric antigen receptor, the nucleic acid molecule, the vector and/orthe immune effector cell in the manufacture of a medicament, wherein themedicament is useful for treating a disease or disorder associated withCD19 expression. The doses of the medicament for administration mayrefer to those defined for the immune effector cell as mentioned above.

In another aspect, the present disclosure further provides a method fortreating a disease or disorder associated with CD19 expression,comprising applying the chimeric antigen receptor, the nucleic acidmolecule, the vector and/or the immune effector cell described herein toa subject in need thereof. According to the present disclosure, theadministration of the composition may be effected by different ways,e.g., by intravenous, intratumor, intraperitoneal, subcutaneous,intramuscular, topical or intradermal administration.

In another aspect, the chimeric antigen receptor, the nucleic acidmolecule, the vector and/or the immune effector cell described herein isuseful for the treatment of a disease or disorder associated with CD19expression.

According to the present disclosure, the medicament may include T-cellimmunotherapy agents.

According to the present disclosure, the disease or disorder associatedwith CD19 expression may include non-solid tumors.

According to the present disclosure, the disease or disorder associatedwith CD19 expression may include leukemia and/or lymphoma.

In certain embodiments, the disease or disorder associated with CD19expression may comprise acute lymphoblastic leukemia (ALL), such asadult acute lymphoblastic leukemia (ALL) and/or childhood acutelymphoblastic leukemia (ALL).

In other embodiments, the disease or disorder associated with CD19expression may comprise adult chronic lymphocytic leukemia (CLL). Inother embodiments, the disease or disorder associated with CD19expression may comprise B-cell lymphoma. For example, the B-celllymphoma may comprise non-Hodgkin's lymphoma.

According to the present disclosure, the subject may comprise a human ornon-human animal. For example, the subject can include, but not limitedto, cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

Without wishing to be bound by any theory, the following examples areonly described to illustrate the chimeric antigen receptor, the immuneeffector cell, the preparation method and use of the present disclosure,and are not used to limit the scope thereof. The examples do not includedetailed descriptions of traditional methods, such as those used toconstruct vectors and plasmids, methods of inserting genes encodingproteins into such vectors and plasmids, or methods of introducingplasmids into host cells. Such methods have been well-known to those ofordinary skill in the art, and described in many publications, includingSambrook, J., Fritsch, E. F. and Maniais, T. (1989) Molecular Cloning: ALaboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press.

All patents, applications and references cited in this disclosure areincorporated herein by reference in their entirety, to the extent thateach document is cited for reference individually. If any materialincorporated by reference contradicts or is inconsistent with thisdescription, the description will supersede any of such materials.

EXAMPLES Example 1. Construction and Preparation of a Lentiviral Vector

A fragment containing the CAR structure described in the presentdisclosure (its amino acid sequence and nucleotide sequence are shown inSEQ ID NO. 1 and SEQ ID NO. 2, respectively) was artificiallysynthesized, and constructed into a modified empty vector (manufacturer:SBI Corporation, catalog number: CD500-CD800). Three helper plasmidswere used for packaging to obtain a lentiviral vector, and the obtainedlentiviral vector was named the expression vector CNCT19. The specificsteps are described as follows.

1.1 Construction of a Plasmid Vector

In the present disclosure, a four-plasmid system containing a targetplasmid (Seq1), PMD2.G, pMDLg-pRRE and pRSV-Rev was used.

Firstly, the target plasmid Seq1 was constructed. An empty vector(manufacturer: SBI System BioScience; name: PCDH-EF1-MCS-T2A-copGFP;catalog number: CD526A-1) was cleaved with the two restriction enzymesNhe I (GCTAGC) and Not I (GCGGCCGC), and was connected to the CAR genefragment which contained Nhe I and Not I enzyme-digested sequences atboth ends and encoded the amino acid sequence shown in SEQ ID NO:1, andthe GFP sequence was removed to obtain the vector {circle around (1)}.The vector {circle around (1)}was cleaved with the two restrictionenzymes Not I (GCGGCCGC) and Sal I (GTCGAC), and the T2A sequence wasremoved. The sticky ends were made blunt with the Klenow Fragmentenzyme, and solution I ligase was added for ligation to obtain thevector {circle around (2)}. A PCR method was performed to amplify thesequence of the vector {circle around (2)} except the ampicillinresistance (AmpR) genes, and amplify the synthetic kanamycin resistance(KanR) gene, and then the two fragments was ligated with a recombinaseto obtain the target plasmid Seq1 which contained the nucleotidesequence shown in SEQ ID NO: 2.

After that, three helper plasmids were prepared, wherein helper plasmid1 was PMD2.G (purchased from Biovector, with a product number ofBiovector 12259); helper plasmid 2 was pMDLg-pRRE (purchased fromBiovector Corporation, with a product number of Biovector012251); andhelper plasmid 3 was pRSV-Rev (purchased from Biovector Corporation,with a product number of Biovector012253).

The four plasmids, Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev, were used forpackaging in the following different four plasmid ratios:

ratio 1 of Seq1: PMD2.G: pMDLg-pRRE: pRSV-Rev was 11.8: 3.53: 6.33: 2.3;

ratio 2 of Seq1: PMD2.G: pMDLg-pRRE: pRSV-Rev was 13.8: 3.48: 5.31:2.54;

ratio 3 of Seq1: PMD2.G: pMDLg-pRRE: pRSV-Rev was 14:4.67:4.67:4.67;

ratio 4 of Seq1: PMD2.G: pMDLg-pRRE: pRSV-Rev was 2:1:1:1;

ratio 5 of Seq1: PMD2.G: pMDLg-pRRE: pRSV-Rev was 7:3:5:5;

ratio 6 of Seq1: PMD2.G: pMDLg-pRRE: pRSV-Rev was 9:3:4:6; and

in the comparative example, the ratio of Seq1:PsPAX2:PMD2.G was14:10.5:3.5.

1.2 Preparation of a Lentiviral Vector

(1) Recovery of cells: cryopreserved 293T/17 cells were recovered andthe recovered cells were cultured at 37° C. with 5% CO₂;

(2) Subculture of cells: the recovered cells were subcultured in T175culture flasks until the cell growth density reached 85-95% of thebottom of the flask;

(3) Cell pretreatment: the cells were added to a 10 cm-culture dish at5×10⁶ cells/dish, and cultured at 37° C. with 5% CO₂;

(4) Preparation of a packaging system: 2 ml of DMEM was taken, thetarget plasmid and three helper plasmids were added in a specific ratioto form a four-plasmid system, 28 μl of PEI was added, and the mixturewas thoroughly mixed and allowed to stand at room temperature for 15min;

(5) Plasmid transfection: cells were pretreated and cultured until thecell growth density reached 60-90% of the bottom of the dish, and theprepared solution of the packaging system was transferred to the culturedish for transfection; the transfection was carried out at 37° C., with5% CO₂;

(6) Collection of viruses: 16-18 h after transfection, the medium waschanged with an equal volume of DMEM containing 10% FBS and theincubation was continued for 24 hours before the virus liquid wascollected for the first time, then the medium was changed and an equalvolume of DMEM containing 10% FBS was added, and another 24 hours ofincubation was performed before the virus liquid was collected for thesecond time, wherein the incubation was performed at 37° C. with 5% CO₂.

(7) Clarification of the virus liquid: the lentiviral vector liquid wascentrifuged at 19° C. at 3000 rpm for 15 min, the precipitate wasdiscarded, and supernatant was obtained;

(8) Concentration of viruses: the supernatant was filtered with a 0.45μm filter to a high-speed centrifuge tube and centrifuged at a highspeed of 50,000 g at 4° C. for 90 min, the supernatant was discarded,the precipitate was dissolved in lymphocyte culture medium before beingstored in a low-temperature refrigerator at −80° C. for later use, and alentiviral vector packaged by a four-plasmid system was obtained andnamed CNCT19 lentiviral vector.

1.3 Determination of Physical Titer and Transfection Titer

The concentrated virus solution was taken at 210 μl/tube, the physicaltiter was determined using ELISA, and the transfection titer wasdetected using a flow cytometer. The physical titer indicates the numberof lentiviral particles. The transfection titer represents the number ofactive lentiviral particles.

The test results are shown in Table 1 and Table 2, and FIG. 1A and FIG.1B. The results of Table 1, FIG. 1A and FIG. 1B show that configurationof the lentivirus packaging systems in the quality ratios of the fourplasmids (Seq1: PMD2.G: pMDLg-pRRE: pRSV-Rev) of 11.8: 3.53: 6.33: 2.3,13.8: 3.48: 5.31: 2.54 and 14: 4.67: 4.67: 4.67 resulted in almost thesame packaging efficiency and all led to higher transfection titer thanthe comparative example. Table 2 shows that the quality ratios of thefour plasmids (Seq1: PMD2.G: pMDLg-pRRE: pRSV-Rev) of 7: 3: 5: 5 and 9:3: 4: 6 led to significantly higher transfection titer than ratio 4 andthe ratio of the comparative example, with the transfection titerreaching two times more than that resulting from ratio 4 and the ratioof the comparative example. The results show that the transfection titerresulting from ratios 1, 2 and 3 was higher than that resulting fromratios 4, 5 and 6 and the ratio of the comparative example, wherein thetransfection titer resulting from ratios 1, 2 and 3 was two times morethan that resulting from ratio 4 and the ratio of the comparativeexample.

TABLE 1 The transfection titer and the physical titer resulting fromratios 1, 2 and 3 Transfec- Phys- Quality ratios of the plasmids tionical No. Seq1:PMD2.G:pMDLg-pRRE:pRSV-Rev titer titer Ratio 111.8:3.53:6.33:2.3  4.2E+07 36.322 Ratio 2 13.8:3.48:5.31:2.54 3.59E+0733.56 Ratio 3 14:4.67:4.67:4.67 4.03E+07 31.948

TABLE 2 The transfection titer and the physical titer resulting fromratios 4, 5, 6 and 7 and the ratio of the comparative example Transfec-Quality ratios of the plasmids tion No. Seq1:PMD2.G:pMDLg-pRRE:pRSV-Revtiter Ratio 4 2:1:1:1 1.47E+07 Ratio 5 7:3:5:5 3.18E+07 Ratio 6 9:3:4:63.33E+07 Ratio of the (Seq1:PsPAX2:PMD2.G) 1.37E+07 comparative14:10.5:3.5 example

Example 2. Preparation of T Cells Infected with Lentiviruses

The infection experiment was carried out according to conventionalmethods known to those skilled in the art. The steps of infection arebriefly described as follows:

1. Sorting of T cells

Peripheral blood mononuclear cells (PBMC) were isolated from thesubject's apheresis cells, and then T cells were sorted from the PBMCcells.

2. Activation of the T cells

The isolated T cells were resuspended with complete lymphocyte culturemedium (Xvivo15 medium+5% FBS+100 IU/ml IL-2 or Xvivo15 medium+5% FBS+20ng/ml IL-21+10 ng/ml IL-7) to give a final concentration of (1˜2)×10⁶cells/ml, and 5 to 10 μl of CD3/CD28 stimulation magnetic beads wereadded. The mixture was well mixed, and placed in an incubator forculture for at least 24 h under the culture condition of 37° C+5% CO₂.

3. Infection of T cells with lentiviruses

The activated cultured T cells were taken out, and polybrene at a finalconcentration of 8 μg/ml was added and mixed well. The CNCT19 lentiviralvector obtained in Example 1 was slowly added at MOI=2. After wellmixed, the mixture was placed in a centrifuge and centrifuged at 1500rpm for 1.5 h. After that, it was placed in an incubator for culture forat least 24 h under the culture condition of 37° C. +5% CO₂.

4. Expansion culture of infected T cells

The infected cells were taken out and the cell density was monitored tokeep it at (0.5˜1)×10⁶ cells/ml for use in subsequent examples. Theobtained infected T cells were named CNCT19 cells (i.e., the immuneeffector cells described herein).

Example 3. Detection of the Expression of CAR Molecules on the Surfaceof CNCT19 Cells

The steps of the experiment are as follows:

(1) The CNCT19 cell suspension was centrifuged at 300 g for 5 min, thesupernatant was discarded, sheath fluid was added to resuspend until theviable cell density reached (0.5˜1)×10⁷ cell s/ml . (2) Two flowcytometry tubes were taken for each sample, and labeled Tube 1 and Tube2; Tube 1 was a blank control and there was no need to add antibodies toit, and to Tube 2 was added 10 μL of a ten-fold dilution of Alexa Fluor®647-goat anti-mouse IgG F(ab′)₂ antibody (manufacturer: Jackson, catalognumber: 115-605-072).

(3) To each tube was added 100 μl of cell suspension, and reaction wasperformed for 15 to 20 min in the dark at room temperature.

(4) To each tube was added 2 ml of sheath fluid for washing, andcentrifugation was performed at 300 g for 5 min.

(5) The supernatant was discarded, and 20 μl of FITC-CD3 (manufacturer:Tongsheng Shidai, catalog number: Z6410047-100T) was added to Tube 2,and incubated for 15 to 20 min in the dark at room temperature.

(6) To each tube was added 2 ml of sheath fluid for washing,centrifugation was performed at 300 g for 5 min, the supernatant wasdiscarded, an additional 2 ml of sheath fluid was added to each tube forwashing, and centrifugation was performed at 300 g for 5 min.

(7) The supernatant was discarded, and after resuspension was performedby adding 300 μl of sheath fluid to each tube, detection was performedby flow cytometry.

The results are presented in FIG. 2A and FIG. 2B. As can be seen, FIG.2A represents a blank control tube; its upper-right CD3+CAR+ quadrantshows no cell population. FIG. 2B represents an experimental detectiontube. CART cells were labeled with IgG F(ab′)₂ and CD3 antibodies and aCAR expression rate of 68.6% can be clearly detected in the CD3⁺CAR+quadrant by flow cytometry. The results indicate that the CAR moleculesaccording to the present disclosure are well expressed on the surface ofCNCT19 cells.

Example 4. Detection of the Killing Effect of CNCT19 Cells on TargetCells In Vitro

The experimental steps of this example are as follows:

1) The CD19-positive human leukemia cell line Nalm-6 (purchased fromShanghai Enzyme Research Bioscience Co., Ltd., catalog number: CH179)and the CD19-negative human leukemia cell line KG-1a (purchased fromShanghai Enzyme Research Bioscience Co., Ltd., catalog number: CC-Y1305)were respectively selected as the tumor cells (i.e., target cells). CD19CAR-T cells (i.e., CNCT19 cells obtained in Example 2) and untransfectedT cells (denoted by NTD) were respectively selected as the effectorcells.

2) The aforementioned target cells and effector cells were mixed ateffector/target ratios of 1:2 and 2:1 respectively and seeded in a24-well plate. The total number of the cells co-cultured in each wellwas made to be about 1×10⁶/well, and three parallel wells were set foreach condition. Each well was replenished with culture solution to 1 ml,the plate was placed in an incubator at 37° C. with 5% CO₂ for culture,and the time was recorded.

3) After 24 hours of co-culture, cell suspension in each well wascollected, transferred to 1.5 mL EP tubes, and labeled respectively. Inaddition, after centrifugation, the supernatant from each sample tubewas sucked into a new 1.5 mL EP tube, and cryopreserved at −20° C. forsubsequent cytokine detection (see Example 6 for details).

4) According to the type of the tumor cells, a corresponding detectionamount of antibodies were respectively added to the mixed cells in eachwell for labeling, and an operation was performed according to theantibody instruction. PE-CD10 antibodies were used to label Nalm-6cells, and Percp-cy5.5-CD45 antibodies were used to label KG-1a cells.

5) Flow cytometry was used to detect the change in the proportion ofdifferent target tumor cells in each sample.

The results are shown in FIG. 3 . As can be seen, compared with theco-culture with untransfected T cells (i.e., NTD), co-culture of theCD19-positive tumor cells Nalm-6 with CAR-T cells (i.e., CNCT19 cells)led to a significantly reduced residual rate of Nalm-6; nevertheless,there was no significant difference in the residual rates of KG-1a afterthe CD19 negative tumor cells KG-1a were co-cultured with variouseffector cells. Specifically, where the effector/target ratio was 1:2,co-incubation of CNCT19 cells with Nalm-6 cells led to a residual rateof target cells of (2.8±1.3)%, which was significantly lower than that((12.1±1.2)% (P<0.01)) resulting from co-incubation of untransfected Tcells with Nalm-6 cells. Likely, where the effector/target ratio was2:1, co-incubation of CNCT19 cells with Nalm-6 cells led to a residualrate of target cells of (1.1±0.1)%, which was significantly lower thanthat ((7.3±1.2)% (P<0.01)) resulting from co-incubation of untransfectedT cells with Nalm-6 cells. Moreover, the killing effect of CNCT19 cellson CD19-positive tumor cells enhanced with the increase of theeffector/target ratio.

Example 5. Real-Time Monitoring of the Killing Function of CNCT19 Cells

The experimental steps are as follows:

(1) The target cells CHO-CD19 were taken out, the culture solution inthe culture flask was aspirated and discarded, the culture flask waswashed once with physiological saline solution, 1 ml of trypsin solutioncontaining EDTA was added, and incubation was performed in a 37° C.incubator for 2 to 6 min before the digestion was stopped. It should benoted that the CHO cells were purchased from Shanghai Enzyme ResearchBioscience Co., Ltd., with a catalog number of CC-Y2110; the molecularsequences of CD19 cells were derived from NCBI, and the molecularsequences of CD19 were constructed into the CHO cells by a method ofmolecular biology, and the target cells CHO-CD19 cell strains wereobtained through screening.

(2) An appropriate amount of culture medium was added to the cultureflask to make the target cells form a cell suspension, and the cellswere made uniform by pipetting. After the concentration of the cellsuspension was counted with a counting plate, the cell suspension wasformulated to a cell concentration of 1×10⁵ cells/ml as required by theexperiments.

(3) 50 μl A of culture medium was added to the wells of E-Plate 16 ofthe RTCA DP system. E-Plate 16 was placed on the RTCA Station. The RTCAsystem would automatically scan (“Scan Plate”) to check whether thecontact was good (“Connection OK” was displayed on the “Message” page).Detection of the baseline (Background) was started to make sure that theselected well was in normal contact.

(4) E-Plate 16 was taken out, and 100 μl of well-mixed target cellsuspension was added to the wells at 1×10⁴ cells per well. The E-Plate16 was placed in a super clean bench at room temperature for 30 min, andthen was placed on the RTCA Station in the incubator. After the systemautomatically scanned (“Scan Plate”), Step 2 was started to dynamicallydetect the cell proliferation curve in real time.

(5) E-Plate 16 was taken out, the target cell suspension and CNCT19 cellsuspension were added to some of the wells, and the target cellsuspension and untransfected T cell (i.e., NTD) suspension (as acontrol) were added to other wells, wherein the effector/target ratio(i.e., the ratio of the effector cells to the target cells, i.e., CNCT19cells: target cells; and NTD: target cells) was 1:1, and the volume ofthe target cell suspension was 50 μl. The E-Plate 16 detection plate wasplaced on the RTCA DP detection platform for 60 h real-time monitoringto observe the effect of CNCT19 cells on the target cells.

The results are shown in FIG. 4 . In the figure, line 1 represents thecurve of the NTD control group, and line 2 represents the curve of theCNCT19 treatment group. By comparing line 1 with line 2, it can be seenthat the proliferation of tumor cells (i.e., target cells) was inhibitedby CNCT19 cells with time. Specifically, after co-incubation withuntransfected T cells, the target cells showed no significant change inthe growth trend (line 1). In contrast, after co-incubation with CNCT19cells, the target cells showed significantly decreased growth trend, andeven started to show a decrease in their number (line 2). This showsthat CNCT19 has strong killing ability against target cells.

Example 6. Detection of Secreted Cytokines after Co-Culture of CNCT19Cells with Target Cells

The experimental steps of this example are as follows:

1) The supernatant sample from the mixed culture in each well in Example4 (i.e., the sample obtained in step 3) was taken out from the −20° C.refrigerator and melted at room temperature;

2) A LEGENDplex™ kit (manufacturer: Biolegned, catalog number: 740013)was used to treat each sample according to the instructions; and 3) Thelevels of different cytokines in each sample were detected by flowcytometry.

The results are shown in FIG. 5A and FIG. 5B. Compared withuntransfected T cells (i.e., NTD), co-culture of CAR-T cells (i.e.,CNCT19 cells) with Nalm-6 cells (CD19⁺) resulted in a significantlyincreased secretion of the cytokines IFN-γ and IL-6 by CAR-T cells.Specifically, after co-cultured with the target cells at aneffector/target ratio of 2:1 for 24 h, CNCT19 cells were stimulated bythe target cells to secrete INF-γ in an amount of (6186.37±861.13)pg/ml, which was significantly higher than the amount of INF-γ(2096.85±228.16 pg/ml, P<0.05) secreted by the untransfected T cells;the amount of IL-6 (32.22±1.46 pg/ml) secreted by CNCT19 cells wassignificantly higher than that (12.23±4.37 pg/ml, P<0.05) secreted bythe untransfected T cells.

Example 7. Detection of Hemolysis and Irritation of CNCT19 Cells

7.1. Test of Hemolysis of CNCT19 Cells In Vitro

The experimental steps of this example are as follows:

1) A total of 7 glass test tubes were used and numbered 1 to 7, and toeach tube was added 2.5 mL of 2% rabbit red blood cell suspension(harvested from New Zealand rabbits, and produced by the ChineseNational Institutes for Food and Drug Control, with a number of qualitycertification of No. 11400500032425).

2) Different doses (0.5 to 0.1 mL) of CNCT19 cells at a concentration of1×10⁷ cells/mL (based on the total number of T cells) were added totubes 1 to 5 which had already contained different doses (2.0 to 2.4 mL)of sodium chloride injection. Meanwhile, 2.5 mL of sodium chlorideinjection (negative control) and 2.5 mL of sterile water for injection(positive control) were added to tube 6 and tube 7, respectively.

3) Each test tube had a total volume of 5.0 mL. The test tubes wereplaced in a 37° C±0.5° C. incubator for incubation for 3 h. Observationwas made as to whether the red blood cells were lysed or aggregated at15 min, 30 min, 45 min, 1 h, 2 h and 3 h after the test tubes wereplaced in the incubator.

The results are shown in FIG. 6A and FIG. 6B. FIG. 6A shows the observedresults of each test tube before shaking for 3 h, and FIG. 6B shows theobserved results of each test tube after shaking for 3 h. It can be seenthat in the negative control tube (No. 6), the supernatant liquid wascolorless and clear, and the red blood cells sank at the bottom of thetube, and they, after shaking, became evenly dispersed; it was judgedthat there was no hemolysis and no aggregation. In the positive controltube (No. 7), the solution was clear and red with no separated layers,and there was no red blood cell residue at the bottom of the tube; itwas judged that there was complete hemolysis. By observing each of thetest tubes to which different doses of CNCT19 cells were added for 3 h,it can be seen that the supernatant liquid in each test tube wascolorless and clear, and the red blood cells sank at the bottom of thetube, and they, after shaking, became evenly dispersed; it was judgedthat there was no hemolysis and no aggregation.

7.2. Test of Vascular Irritation upon Intravenous Infusion of CNCT19Cells

The experimental steps of this example are as follows:

1) Six New Zealand rabbits (with three males and three females) that hadpassed the quarantine inspection and had no abnormalities in theinjection sites were selected. A self-control method was used:cryopreserved CAR-T cells (i.e., CNCT19 cells) and a negative control(sodium chloride injection) were respectively intravenously infused intothe right and left ears of each animal.

2) The CAR-T cells infused through the right ear-marginal veins were ata concentration of 1×10⁷ cells/mL, and had a dosage of 1×10⁷ cells/kg(based on the total number of T cells). The sodium chloride injectioninfected through the left ear-marginal veins was used as a negativecontrol. The dosage intravenously administered through both the left andthe right ear-marginal veins was 1 mL/kg.

3) After intravenous administration, general observation, observation ofthe injection sites, and pathological examination of the animals werecarried out.

The results are shown in FIG. 7A and FIG. 7B. FIG. 7A shows a micrograph(HE-stained, 10×objective lens) of the injection site after theadministration of CAR-T cells, and FIG. 7B shows a micrograph(HE-stained, 10×objective lens) of the injection site after theadministration of sodium chloride injection. It can be seen that afterintravenous infusion of the cryopreserved CAR-T cells, compared with thenegative control side, no systemic or local symptoms and pathologicalabnormalities of the animals were found.

Example 8. Experiment of Oncogenicity of CNCT19 Cells In Vitro

The experimental steps of this example are as follows:

1) CAR-T cells (i.e., CNCT19 cells) from two donors were respectivelyincubated in soft agar medium. The two donors were Donor 1 (healthyhuman donor T Cells, lot number: TC20180613015) and Donor 2 (healthyhuman donor T Cells, lot number: TC20180613016), respectively. Inaddition, the human embryonic lung fibroblast cell line MRC-5 was set asa negative control, and the human cervical cancer cell line Hela was setas a positive control.

2) A 6-well culture plate was used in the experiment. The number ofcells in each well was about 1×10³, and three parallel wells were setfor each group. The culture plate was placed in a CO₂ incubator forculture and observation was performed for 3 weeks.

3) The culture plate was taken out every 1 week to observe whether therewas formation of clones with a microscope and take pictures. Theexperiment stopped until formation of obvious clone in the positivecontrol group.

The results are shown in FIG. 8 . It can be seen that the positivecontrol cells (Hela) formed clones in the culture medium, and the sizeof the clones increased significantly with time. On the 23rd day ofobservation, CAR-T cells from different donors and negative controlcells (MRC-5) did not exhibit clonal growth, and they all died, notbeing characterized by immortalized proliferation in vitro.

Example 9. Experiment of Tumorigenicity of CNCT19 Cells In Vivo

The experimental steps of this example are as follows:

1) BALB/c nude mice were subcutaneously inoculated with cryopreservedCAR-T cells (i.e., CNCT19 cells) from two donors, and correspondinguntransfected T cells (i.e., NTD), respectively, wherein the two donorswere Donor 2 (healthy human donor T cells, lot number: TC20180613016)and Donor 3 (healthy human donor T cells, lot number: TC20180808019).

2) Inoculation with CAR-T cells was performed at 1×10⁷ cells/mouse(based on the total number of T cells). The negative control group wasinoculated with 1×10⁷ human embryo lung fibroblast cell line MRC-5 cellsper mouse, and the positive control group was inoculated with 1×10⁶human cervical cancer cell line Hela cells per mouse.

3) Continuous observation was performed for 16 weeks to detect whetherthere was a change in body weight, whether there were nodules generated,and whether the nodules would be induced to become tumorous nodules. Theresults were compared with those of the negative and positive cellgroups.

4) After observation, gross anatomy was performed. Each organ wasweighed, and the organ coefficient was calculated. The inoculation sitesand tissue or organs with suspicious symptoms were subjected tohistopathologic examination.

The results show that CAR-T cells are not tumorigenic in vivo. In thenegative control group (inoculated with MRC-5 cells), liquid nodules ofall animals disappeared by the 9th day after inoculation. In thepositive control group (inoculated with HeLa cells), the subcutaneousnodules of all animals slowly increased. Pathological examination showedthat those nodules were caused by the growth of tumor tissue, with atumor-forming rate of 100%. In summary, this experiment was valid. Inthe group inoculated with CAR-T cells (derived from two donors) anduntransfected T cells, all the liquid nodules disappeared on the 5th dayafter inoculation, and no nodule formed again before euthanasia wasperformed on the 114th day. Pathological examination showed that therewas no tumor formed at the sites of inoculation or metastasis.

Example 10. Test of Toxicity of Single Intravenous Injection of CNCT19Cells to NCG Mice with Nalm-6 Xenograft Tumors

The experimental steps of this example are as follows:

1) NCG mice aged 6-8 weeks were used, wherein half of them were male.Three days before the administration, Nalm-6 cell suspension wasinjected into the tail veins at a concentration of 2.5×10⁶ cells/mL andat a dose of 10 mL/kg.

2) The screened animals were randomly divided into 4 sex-balanced groupsaccording to their body weight (i.e., groups 2 to 5). Groups 2 to 5 werea vehicle control group, a T cell control group, and a CAR-T celllow-dose group and CAR-T cell high-dose group respectively, with 40animals (20 males and 20 females) in each group. Both thenon-tumor-bearing control group (i.e., group 1) and the vehicle controlgroup (i.e., group 2) were given a vehicle control (i.e., physiologicalsaline containing 4% (W/V) human albumin). The T cell control group(i.e., group 3) was given untransfected T cells (i.e., NTD) at a dose of1×10⁹ cells/kg (based on the total number of T cells, the samehereinafter). The CAR-T cell low-dose group (group 4) was inoculated ata dose of 1×10⁸ cells/kg and the CAR-T cell high-dose group (group 5)was inoculated at a dose of 1×10⁹ cells/kg.

3) The dosage for each animal was 25 mL/kg, the administration wasperformed by a single intravenous injection, and the rate ofadministration was about 1 mL/min.

4) After the administration, observation was continuously performed for4 h on the day of administration. During the test, general clinicalobservation was carried out once in the morning and once in theafternoon each day. Detailed clinical observation and measurement ofbody weight and food intake were performed once a week. During the test,the body temperature, clinicopathological indicators (blood cell countand blood biochemical indexes) and immunological indicators (Tlymphocyte subsets, cytokines and C-reactive protein) were detected.

5) In groups 1-5, 10 animals/sex/group were euthanized on day 2 and 5animals/sex/group were euthanized on day 15. Their organs were weighedand gross anatomy observation was performed. The main organs of animalsin groups 1 and 5 were subjected to histopathological examination.

The results of this experiment show that the maximum tolerated dose ofCAR-T cells (i.e., CNCT19 cells) for a single administration was greaterthan 1×10⁹ cells/kg. During the test, none of the animals in the CAR-Tcell low-dose group and CAR-T cell high-dose group was dead or dying. Noabnormal reaction was observed in either the general or the detailedclinical observation. No obvious abnormal changes in the body weight,food intake, body temperature, C-reactive protein, and blood biochemicalindexes were observed. The histopathological examination found noobvious abnormality in the CAR-T cell low-dose group and CAR-T cellhigh-dose group as compared with the T cell control group.

Example 11. Treatment of Animals with CNCT19 Cells

11.1. Therapeutic Effect of CNCT19 Cells on NCG Mice with Nalm-6Xenograft Tumors

The experimental steps of this example are as follows:

1) Female NCG mice aged 6-8 weeks were used. Three days before theadministration, 5×10⁵ Nalm-6 cells were injected into the tail veins,wherein the Nalm-6 cells were dissolved in physiological saline at aconcentration of 2.5×10⁶ cells/ml, and each mouse was injected with 200μl of cell resuspension.

2) Each experimental group was injected with corresponding CAR-T cells(i.e., CNCT19 cells), untransfected T cells (i.e., NTD) or cellpreservation solution (i.e., physiological saline containing 4% (W/V)human albumin) A total of five groups were divided as follows: CAR-Tlow-dose group (injected with CNCT19 cells at 5×10⁶ cells/mouse based onthe total number of T cells, the same hereinafter), CAR-T medium-dosegroup (injected with CNCT19 cells at 1×10⁷ cells/mouse), CAR-T high-dosegroup (injected with CNCT19 cells at 2×10⁷ cells/mouse), T cell controlgroup (injected with untransfected T cells at 2×10⁷ cells/mouse) andvehicle control group (injected with cell preservation solution at 200μl mouse). The injection volume per mouse was 200 μl.

3) The body weight was detected and general clinical observation wasperformed twice a week. The survival of the mice was recorded andsurvival curves were plotted.

The results are shown in FIG. 9 . As can be seen, CNCT19 cells at alldoses have significant effects on prolonging the survival time and theseeffects are obviously dose-dependent. Specifically, the median survivaltime of the physiological saline control group was 24 days, that of theNTD control group was 23 days, and that of the CNCT19 low-dose group was40 days, whereas all the experimental animals in the CNCT19 medium-dosegroup and the CNCT19 high-dose group survived to the end of theobservation period. Compared with the physiological saline group and NTDcontrol group, all the dose groups of CNCT19 can prolong the survivaltime of animals with leukemia by more than 16 days.

11.2. Distribution of CNCT19 Cells in Animals

The experimental steps of this example are as follows:

1) NCG mice aged 6-8 weeks were used, wherein half of them were male.Nalm-6 xenograft tumor models were established by the method in Example11.1.

2) Both tumor-bearing and non-tumor-bearing animals received a singletail vein injection of CAR-T cells (i.e., CNCT19 cells) at a dose of5×10⁶ cells/mouse (based on the total number of T cells).

3) Animals in the tumor-bearing group were euthanized as planned at 24hours (i.e., D2), 72 hours (i.e., D4), 168 hours (i.e., D8), 336 hours(i.e., D15), 504 hours (i.e., D22), and 672 hours (i.e., D29)respectively after the administration. The animals in thenon-tumor-bearing group were euthanized as planned at 24 hours (i.e.,D2), 168 hours (i.e., D8), and 336 hours (i.e., D15) respectively afterthe administration. Animal whole blood (EDTA anticoagulation), brain,spinal cord (cervical segment), skeletal muscle, gonads (ovaries, testesand epididymis), bladder, stomach, small intestine, mesenteric lymphnodes, bone marrow, liver, kidney, spleen, heart, lung and other tissuesor body fluids were collected in order.

4) The content of chimeric antigen receptors (i.e., CARs) in blood andvarious tissue samples was determined using a validated Q-PCR method.

The results show that after being administered to tumor-bearing mice andnon-tumor-bearing mice by a single intravenous injection at a dose of5×10⁶ cells/mouse, the CNCT19 cells were mainly distributed in the wholeblood and tissue with large blood flow such as lung, liver, heart, andspleen (see FIG. 10 ). Among them, heart and the whole blood had thelargest distribution, with the areas under the areas under curve (thecopy number of the nucleic acid molecule encoding CAR in gDNA-time, AUC)being about 150,000 hours*copy number/μg. The lung and spinal cord werefollowed, with AUC being about 40,000 to 60,000 hours*copy number/μg. Asto spleen, liver and other tissue, AUC were less than about 20,000hours*copy number/μg.

The results also show that the content of CNCT19 cells in the tissue oftumor-bearing mice was slightly higher than that of non-tumor-bearingmice (see FIG. 11 ). The concentrations of CNCT19 cells in the wholeblood, heart and spinal cord of tumor-bearing mice were about 3 times, 6times, and 5 times more than those in non-tumor-bearing mice 24 hoursafter administration.

Thereafter, the drug content in each tissue gradually decreased, andbasically fell below the methodological detection limit two weeks afterthe administration. As the disease progressed, with enhancing CD19antigen stimulation, the CNCT19 cells in the mice reactivelyproliferated again (see FIG. 12 ), wherein the concentration of CNCT19cells in the brain, lung, liver, whole blood, and spinal cord increasedto more than 1200 copies/₁1g DNA, and even reached 3500 copies/μg DNA.

Example 12. Treatment of Acute Lymphoblastic Leukemia with CNCT19 Cells

12.1. Clinical use of CNCT19 Cells

The clinical application process is shown in Table 3, and the specificexperimental steps in some stages are described below.

TABLE 3 The process of clinical application of CNCT19 cells Visit DateDescription Screening Week −6 to Patients are screened period week −4 ↓Started in week −4 PBMCs are collected by apheresis from thesuccessfully screened subjects to prepare CNCT19 cell suspension, andthe cell preparation takes 3-4 weeks ↓ Started on day −5 The safetyindexes are checked before the lymph node dissection pretreatment ↓ Thesubjects are subjected to lymph node dissection pretreatment ↓ Startedon day −1 Baseline data are acquired ↓ Treatment On day 0 After thesafety indexes are period checked, the cells are reinfused ↓ On days 2,4, 7, 10, A safety test is performed, and 14 and 21 subjects are visitedand examined and evaluated according to the research flow chart ↓ On day28 After completion of the treatment, an examination is performed, andthe subjects are visited and examined and evaluated according to theresearch flow chart ↓ Follow-up Performed once a The subjects arevisited and examined period month for 6 months and evaluated accordingto the research flow chart ↓ Follow-up for the The subjects are visitedand examined recurrence-free and evaluated according to the survival wasresearch flow chart performed once every three months Follow-up for theFollow-up can be completed by overall survival telephone to obtaininformation on was performed the survival status of the subjects onceevery three and their anti-tumor treatment months for 2 years

1. Lymph node dissection pretreatment

Lymph node dissection pretreatment is performed on day −5 before theCNCT19 cell suspension is reinfused as planned.

The pretreatment schemes are as follows:

Fludarabine is administered at 30 mg/m² once per day for 2-4 consecutivedays; and cyclophosphamide is administered at 500 mg/m² once per day for2 consecutive days.

The use of the two drugs for the pretreatment chemotherapy should bestarted from the same day.

2. Reinfusion of CNCT19 cell suspension

(1) After the cells are successfully prepared, they are cryopreservedunder the condition of <-100° C., transported to the hospital under thesame temperature condition for use, and recovered according to theexperiment operation guide before infusion.

(2) The method for reinfusing cells is as follows. The cells arerecovered according to the experiment operation guide, and thereinfusion of the cell suspension should be complete within 30 minutesafter the recovery. The cell suspension is infused as a single infusioninto the subject through a vein with a blood transfusion device. Ifthere is more than one bag of cell suspension, they can be continuouslyinfused without time interval between reinfusion of each bag. Thesubject needs to be closely observed during the cell reinfusion. If aserious adverse event occurs, the infusion should be stopped, andcorresponding treatment should be performed according to the specificadverse event. If no serious adverse event occurs, follow-up can becarried out in accordance with the visit workflow.

(3) The subject needs to be closely observed for 24 h after the cellreinfusion. If a serious adverse event occurs, corresponding treatmentshould be performed according to the specific adverse event. If noserious adverse event occurs, follow-up can be carried out in accordancewith the visit workflow.

(4) After the cell reinfusion, the subject needs to continue to behospitalised for observation for 14 days or for a duration determinedaccording to the researcher's comprehensive assessment of the subject'scondition.

3. Management of CNCT19 cell suspension

In order to strictly manage and use the CNCT19 cell suspension, a strictsystem of managing CNCT19 cell suspension by specifically assignedpersonnel has been established. Specific personnel are assigned totransport the cell suspension for research to the hospital departments,and specific personnel are assigned responsibility for the reception ofthe cell suspension for research and the establishment of a registrationsystem.

After the infusion, the packaging of the cell suspension is recoveredand stored/destroyed by the drug management personnel.

4. The clinical efficacy and safety results of CNCT19 cell suspension inthe treatment of relapsed or refractory acute lymphoblastic leukemia

This experiment started from September 2016 to October 2020. During theexploratory and phase I clinical trials, 63 patients with relapsed orrefractory acute lymphoblastic leukemia (ALL) (with 23 adult patientsand 40 children patients) were treated with CNCT. The explored doseadministered for adult ALL ranged from 0.25×10⁸ to 0.5×10⁸ CAR-positiveT cells.

(a) The data on the clinical efficacy are shown in Table 4 and Table 5.Table 4 shows the recovery of 63 patients including adults and children.Table 5 shows the recovery of the 23 adult patients. It can be seen thatamong the 63 patients with relapsed or refractory acute lymphoblasticleukemia, after the injection and reinfusion of the CNCT19 cellsuspension, the vast majority (93.7%) of the patients were in completeremission, and 88.9% of the patients were MRD negative. The results showthat injection and reinfusion of the CNCT19 cell suspension caneffectively treat adult patients and children patients with relapsed orrefractory acute lymphoblastic leukemia.

TABLE 4 The clinical efficacy of the CNCT19 cell suspension on treatingacute lymphoblastic leukemia in adults and children Adults and childrenwith relapsed or refractory acute lymphoblastic leukemia N = 63 ORR93.7% (59/63) CR/Cri 93.7% (59/63) MRD negative 88.9% (56/63)

TABLE 5 The clinical efficacy of the CNCT19 cell suspension on treatingacute lymphoblastic leukemia in adults Adults with relapsed orrefractory acute lymphoblastic leukemia N = 23 ORR91.3% (21/23) CR/Cri91.3% (21/23) MRD negative 86.9% (20/23)

b) The data of the preliminary safety results are shown in Table 6 andTable 7. Table 6 shows the safety results for 63 patients includingadult and children patients, and Table 7 shows the safety results for 23adult patients. Among the 63 ALL patients, the incidence of grade 3 andabove CRS and that of encephalopathy were 19% and 20.6%, respectively. Acomparison between the age groups reveals that the incidence (39.1%) ofsevere CRS in the adult group is higher than that (7.5%) in the childgroup, and the incidence (17.4%) of severe CRES in the adult group isslightly lower than or close to that (22.6%) in the child group. Thismay be due to the fact that in the early trials CNCT19 was givenaccording to body weight, and the adult patients with larger body weightreceived higher doses. For example, the dose given to the 46 subjects inthe early trials ranged from 0.71×10⁶ to 4.08×10⁶/kg, equivalent to0.16×10⁸-2.36×10⁸ CAR-positive T cells. As the initial dose-explorationgradually shed light on the characteristics of CNCT19 products, a saferdose range was selected in subsequent clinical studies. For example,among the 17 patients receiving a dose ranging from 0.2×10⁸ to 1.1×10⁸CAR-positive T cells (median value: 0.5×10⁸), the incidence of grade ≥3CRS and CRES dropped to 5.9% (1/17).

It can be seen that injection of the CNCT19 cell suspension has a lowprobability of causing severe CRS or CRES side effects, its overallsafety is controllable, and thus CNCT19 has good safety performance. Thegood safety performance of CNCT19 improves the quality of the productsand reduces the clinical risks.

TABLE 6 The safety results of the CNCT19 cell suspension for adults andchildren with acute lymphoblastic leukemia Cytokine ReleaseCAR-T-cell-related Encephalopathy Syndrome (CRS) Syndrome (CRES) Allgrades Grade ≥3 All grades Grade ≥3 Adults + Total 54 (85.7%) 12 (19%)22 (34.9%) 13 (20.6%) children with (n = 63) acute lymphoblasticleukemia

TABLE 7 The safety results of the CNCT19 cell suspension for adults withacute lymphoblastic leukemia Cytokine Release CAR-T-cell-relatedEncephalopathy Syndrome (CRS) Syndrome (CRES) All grades Grade ≥3 Allgrades Grade ≥3 Adults with Total 20 (86.9%) 9 (39.1%) 8 (34.8%) 4(17.4%) acute (n = 23) lymphoblastic leukemia Note: in Tables 6 and 7,CRS stands for cytokine release syndrome; and CRES stands forCAR-T-cell-related encephalopathy syndrome.

Example 13. Treatment of Relapsed or Refractory Non-Hodgkin's Lymphomawith CNCT19 Cells

13.1. Clinical use of CNCT19 Cells

The clinical application process is shown in Table 8, and the specificexperimental steps in some stages are described below.

TABLE 8 The process of clinical application of CNCT19 cells Visit DateDescription Screening Week −8 to the time Patients are screened periodbefore the apheresis ↓ Week −7 to day −1 PBMCs are collected byapheresis from the successfully screened subjects to prepare CNCT19 cellsuspension, and the cell preparation takes 3-4 weeks ↓ Day −5 to day −2The safety indexes are checked before the lymph node dissectionpretreatment ↓ The subjects are subjected to lymph node dissectionpretreatment ↓ Started on day −1 Baseline data are acquired ↓ TreatmentOn day 0 After the safety indexes are checked, period the cells arereinfused ↓ On days 2, 4, 7, 10, A safety test is performed, and 14 and21 subjects are visited and examined and evaluated according to theresearch flow chart ↓ On day 28 After completion of the treatment, anexamination is performed, and the subjects are visited and examined andevaluated according to the research flow chart ↓ Follow-up Follow-up forthe The subjects are visited and examined period overall remission andevaluated according to the rate was performed research flow chart once amonth for 6 ↓ months Follow-up for the The subjects are visited andexamined recurrence-free and evaluated according to the survival wasresearch flow chart performed once every three months Follow-up for theFollow-up can be completed by overall survival telephone to obtaininformation on was performed the survival status of the subjects onceevery three and their anti-tumor treatment months for 2 years

1. Lymph node dissection pretreatment

Lymph node dissection pretreatment is performed on day −5 before theCNCT19 cell suspension is reinfused as planned.

The pretreatment schemes are as follows:

Fludarabine is administered at 30 mg/m² once per day for 2-4 consecutivedays; and cyclophosphamide is administered at 500 mg/m² once per day for2 consecutive days.

The use of the two drugs for the pretreatment chemotherapy should bestarted from the same day.

2. Reinfusion of CNCT19 cell suspension

(1) After the cells are successfully prepared, they are cryopreservedunder the condition of <-100° C., transported to the hospital under thesame temperature condition for use, and recovered according to theexperiment operation guide before infusion.

(2) The method for reinfusing cells is as follows. The cells arerecovered according to the experiment operation guide, and thereinfusion of the cell suspension should be complete within 30 minutesafter the recovery. The cell suspension is infused as a single infusioninto the subject through a vein with a blood transfusion device. Ifthere is more than one bag of cell suspension, they can be continuouslyinfused without time interval between reinfusion of each bag. Thesubject needs to be closely observed during the cell reinfusion. If aserious adverse event occurs, the infusion should be stopped, andcorresponding treatment should be performed according to the specificadverse event. If no serious adverse event occurs, follow-up can becarried out in accordance with the visit workflow.

(3) The subject needs to be closely observed for 24 h after the cellreinfusion. If a serious adverse event occurs, corresponding treatmentshould be performed according to the specific adverse event. If noserious adverse event occurs, follow-up can be carried out in accordancewith the visit workflow.

(4) After the cell reinfusion, the subject needs to continue to behospitalised for observation for 14 days or for a duration determinedaccording to the researcher's comprehensive assessment of the subject'scondition.

3. Management of CNCT19 cell suspension

In order to strictly manage and use the CNCT19 cell suspension, a strictsystem of managing CNCT19 cell suspension by specifically assignedpersonnel has been established. Specific personnel are assigned totransport the cell suspension for research to the hospital departments,and specific personnel are assigned responsibility for the reception ofthe cell suspension for research and the establishment of a registrationsystem.

After the infusion, the packaging of the cell suspension is recoveredand stored/destroyed by the drug management personnel.

4. The clinical efficacy and safety results of CNCT19 cell suspension inthe treatment of relapsed or refractory non-Hodgkin's lymphoma

This experiment started from September 2016 to October 2020. During theexploratory and phase I clinical trials, 50 patients with relapsed orrefractory non-Hodgkin's lymphoma (NHL) were treated with CNCT19 cellsuspension. The explored dose administered for NHL (in adults only)ranged from 1×10⁸ to 2×10⁸ CAR-positive T cells.

(a) The data on the clinical efficacy are shown in Table 9. Table 9shows the recovery of 50 patients. It can be seen that among the 50patients with relapsed or refractory non-Hodgkin's lymphoma, after theinjection and reinfusion of the CNCT19 cell suspension, about 80% of thepatients were in complete remission or partial remission, wherein therate of complete remission was 54% (27 cases) and the rate of partialremission was 24% (12 cases). This shows that the CNCT19 cell suspensioncan effectively treat patients with relapsed or refractory non-Hodgkin'slymphoma.

TABLE 9 The clinical efficacy of the CNCT19 cell suspension on treatingnon-Hodgkin's lymphoma Relapsed or refractory non-Hodgkin's lymphoma N =50 ORR 78% (39/50) CR 54% (27/50) PR 24% (12/50) Note: in Table 9, ORRstands for overall remission rate; CR stands for complete remission; andPR stands for partial remission.

b) The data of the preliminary safety results are shown in Table 10.Table 10 shows the safety results for 50 patients. It can be seen thatinjection of the CNCT19 cell suspension has a low probability of causingsevere CRS or CRES side effects, and has a probability of causing Grade≥3 side effects of lower than 10%, wherein the probability of causingGrade ≥3 CRS is 0% and the probability of causing Grade ≥3 CRES is 6%.Thus, CNCT19 has good safety performance.

TABLE 10 The safety results of the CNCT19 cell suspension fornon-Hodgkin's lymphoma Cytokine Release CAR-T-cell-relatedEncephalopathy Syndrome (CRS) Syndrome (CRES) All grades Grade ≥3 Allgrades Grade ≥3 Non-Hodgkin's Total 30 (60%) 0 (0) 3 (6%) 3 (6%)lymphoma (n = 50) Note: in Table 10, CRS stands for cytokine releasesyndrome; and CRES stands for CAR-T-cell-related encephalopathysyndrome.

The foregoing detailed description is provided by way of explanation andexample, but is not intended to limit the scope of the attached claimed.The various changes to the embodiments enumerated in the disclosure areapparent to one of ordinary skill in the art and are retained within thescope of the attached claims and equivalents thereof.

1. A plasmid combination, wherein the plasmid combination comprisesplasmids Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev; and the plasmids Seq1,PMD2.G, pMDLg-pRRE and pRSV-Rev are present at a ratio of 2-6: 1-1.5:1-3: 1-1.5; wherein the plasmid Seq1 is a plasmid comprising a nucleicacid molecule encoding a chimeric antigen receptor.
 2. The plasmidcombination according to claim 1, wherein the plasmid Seq1 comprises anucleic acid molecule encoding the chimeric antigen receptor, whereinthe chimeric antigen receptor comprises an amino acid sequence shown inSEQ ID NO.
 1. 3. The plasmid combination according to claim 1, whereinthe plasmid Seq1 comprises a nucleic acid molecule encoding a chimericantigen receptor, wherein the nucleic acid molecule comprises a nucleicacid sequence shown in SEQ ID NO.
 2. 4. The plasmid combinationaccording to claim 1, wherein the plasmids Seq1, PMD2.G, pMDLg-pRRE andpRSV-Rev are present at a ratio of 3-6: 1.5: 1-3: 1-1.5.
 5. A method forpreparing a lentiviral vector, the method comprising introducing theplasmid combination according to claim 1 into a cell.
 6. The methodaccording to claim 5, wherein the cell is 293T.
 7. A method forpreparing a modified immune effector cell, comprising preparing thelentiviral vector according to the method according to claim
 5. 8. Themethod according to claim 7, comprising introducing the lentiviralvector into an immune effector cell.
 9. The method according to claim 8,wherein the immune effector cell is selected from the group consistingof a T lymphocyte and a natural killer cell.
 10. A modified immuneeffector cell prepared by the method according to claim
 7. 11.(canceled)
 12. A composition comprising the modified immune effectorcell according to claim
 10. 13-20. (canceled)
 21. A method for treatinga disease or disorder associated with CD19 expression, comprisingadministrating one or more of the following to a patient in needthereof: the immune effector cell according to claim 10, and acomposition comprising the modified immune effector cell according toclaim
 10. 22. The method according to claim 21, wherein the disease ordisorder associated with CD19 expression comprises non-solid tumors. 23.The method according to claim 22, wherein the non-solid tumor comprisesleukemia and/or lymphoma.
 24. The method according to claim 21, whereinthe disease or disorder associated with CD19 expression comprises acutelymphoblastic leukemia and/or B-cell lymphoma.
 25. The method accordingto claim 24, wherein the acute lymphoblastic leukemia comprises acutelymphoblastic leukemia in adults and/or acute lymphoblastic leukemia inchildren.
 26. The method according to claim 25, wherein the step ofadministrating is administered at a dose of 0.25×10⁸ to 0.5×10⁸CAR-positive T cells.
 27. The method according to claim 24, wherein theB-cell lymphoma comprises non-Hodgkin's lymphoma.
 28. The methodaccording to claim 27, wherein the step of administrating isadministered at a dose of 1×10⁸ to 2×10⁸ CAR-positive T cells. 29-32.(canceled)
 33. The method according to claim 25, wherein the acutelymphoblastic leukemia is relapsed or refractory acute lymphoblasticleukemia.
 34. (canceled)
 35. The method according to claim 27, whereinthe non-Hodgkin's lymphoma is relapsed or refractory non-Hodgkin'slymphoma.
 36. (canceled)
 37. A plasmid combination, wherein the plasmidcombination comprises plasmids Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev;and the plasmids Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev are present at aratio of 11.8: 3.53: 6.33: 2.3; 13.8: 3.48: 5.31: 2.54; or 14: 4.67:4.67: 4.67.